The field of analytical and diagnostic chemistry has grown tremendously in recent years. The field relates to analysis of body fluids, such as blood, serum, urine and the like, food substances, such as milk, drinking water, and other fluid substances. Applications of the field include medical diagnosis, purity testing, forensic science, etc.
The apparatus employed in analytical chemistry of the type discussed herein is generally a device referred to at times as a test strip, a dip stick, or other terms which will be familiar to those skilled in the art. The device contains various substances incorporated therein, which react with a particular analyte, or substance to be determined, with formation of a determinable or detectable signal. Frequently, but not always, this is a color. Color formation, or change in color, permits one to give a "yes-no" answer to the question of whether a substance or analyte is present in a test sample. Degree of color formed, or the actual color, can be used as an indication of how much analyte is present.
In practice, the sample may be applied to the test device, or the device may itself be dipped in or otherwise contacted to the test sample.
One very common test which is presented as exemplary of the type of assay performable with test devices and dry chemistry is a determination of blood glucose level. This, of course, is important in many situations, such as monitoring of diabetic and hypoglycemic patients. A test strip is prepared which contains the enzymes glucose oxidase and a peroxidase, as well as the indicator 3,3',5,5'-tetramethylbenzidine, also referred to as "TMB". If glucose is present in the sample, it reacts with oxygen in the presence of glucose oxidase to form gluconic acid and hydrogen peroxide. In the presence of peroxidase the peroxide, in turn, reacts with the TMB. TMB, in unoxidized form, is colorless. When oxidized, it forms a color in the range of blue to purple. Thus, one can determine if glucose is present, and in what concentration, by observing formation of color. The system is analyte specific, because hydrogen peroxide is not a normal component of the sample, and only forms when the glucose specific enzyme glucose oxidase acts on its substrate.
The system described supra, is, of course, only one example of the various assays which can be performed using dry chemistry techniques. By modifying the analyte specific enzyme and indicator, one can, of course, determine different analytes, using the system outlined.
Many different types of indicators are available to the clinical chemist. A broad class of such indicators, or indicator systems, rely on the oxidative coupling of two different components to produce an indicator or dye. Exemplary of such systems is the so-called "Trinder reagent", as taught by Trinder, Ann. Clin. Biochem 6:24 (1969). This system is used to determine glucose, cholesterol, and uric acid in body fluids such as serum and plasma.
Trinder reagents involve the coupling of substituted or unsubstituted phenols with 4-aminoantipyrine (4-AAP), to form a red dye. This reaction: ##STR1## is described in Acquati, et al., U.S. Pat. No. 4,291,121, the disclosure of which is incorporated by reference herein. Acquati, et al., teach that various substituted phenols, can be used as the coupling compound for the indicator system.
Acquati, et al. also point to a problem in indicator systems, i.e., interference by components of the sample being tested. The interference in Acquati, et al., results in competition between the compound being determined (cholesterol, glucose, uric acid, etc.), and reducing agents for the indicator system and lower values for the analyte than actually exist.
Klose, et al., in U.S. Pat. No. 4,101,381 teach another type of system: that of the indicator system which produces a charge carrying or "reducible" chromogen. This patent teaches that the compound 3-methyl-2-(sulfonyl)-benzothiazolone hydrazone (MBTHS), reacts in the presence of formaldehyde and an oxidizer to produce a reducible chromogen.
Other indicators such as 3,3',5,5' tetramethylbenzidine (TMB) and its derivatives form charge carrier complexes rather than "coupled oxides" in the manner of Trinder reagents. The charge carrier complexes also serve as indicators in that, when the molecules are not complexed they either carry no color or are a different color from that of the complex.
When these indicators are reduced, however, they do not produce a color. Hence, they are reducible chromogens. Examples, in addition to TMB and MBTH are o-dinisidine, guaicol, and o-tolidine.
This distinction between charge carriers and couplers has been described as well by Katsuyama, et al., U.S. Pat. No. 4,418,037, who describe the distinction as being between "reducible chromogens" and "combination of a hydrogen donor (developing agent) and a coupler". Katsuyama, et al. teach that there is a problem in indicator systems in that enzymes used therein frequently become inactivated, slowing down the reaction producing the signal. In order to avoid this, they propose the addition of pyrogallol derivatives to systems which in their oxidized state "couple with a coupler such as naphthol, a phenol, a pyrazoline or N,N-disubstituted anilines" (column 6, lines 54-60). All of Katsuyama, et al.'s examples require coupling compounds.
Katsuyama teaches that the pyrogallol derivative acts as a "preservative" for the enzyme in the test system. Expressed another way, this patent teaches that in systems using coupling indicators, the shelf-life of the strip is increased. The indicator reaction takes places in the same way it would were the pyrogallol derivative not present.
It has now been found, however, that pyrogallol derivatives, when used in connection with reducible chromogens act in a manner not expected from Katsuyama. It has been found that indicator systems using reducible or charge carrying chromogens function differently "after" addition of pyrogallol than they do before. This is significant because the addition of pyrogallol derivatives to the aforementioned indicator system extends the range of such systems. This will be explained in more detail infra.
Hence it is an object of this invention to provide a composition useful in determining an analyte in a sample, which contains an indicator system characterized by a reducible charge carrier forming chromogen, and a pyrogallol derivative.
It is a further object of the invention to provide kits which can be used to detect analytes in samples, which kits include the above referenced components.
It is a further object of the invention to provide analytical apparatus which can be used to determine analytes, which also use the above identified compositions and kits.
It is still another object of the invention to provide a method for the determination of an analyte in a sample using the above compositions, kits, and analytical apparatus.
How these and other objects of the invention are achieved will become apparent after review of the disclosure which follows.